title: Biochemical characterization of the catalytic domains of three different Clostridial collagenases.
abstract: Clostridial collagenases are used for a broad spectrum of biotechnological applications and represent prime target candidates for both therapy and diagnosis of clostridial infections. In this study, we biochemically characterized the catalytic domains of three clostridial collagenases, collagenase G (ColG) and H (ColH) from Clostridium histolyticum, and collagenase T (ColT) from C. tetani. All protein samples showed activity against a synthetic peptidic substrate (furylacryloyl-Leu-Gly-Pro-Ala, FALGPA) with ColH showing the highest overall activity and highest substrate affinity. Whereas the K(m) values of all three enzymes were within the same order of magnitude, the turnover rate k(cat) of ColG decreased 50- to 150-fold when compared to ColT and ColH. It is noteworthy that the protein N-terminus significantly impacts their substrate affinity and substrate turnover as well as their inhibition profile with 1,10-phenanthroline. These findings were complemented with the discovery of a strictly conserved double-glycine motif, positioned 28 amino acids upstream of the HEXXH zinc binding site, which is critical for enzymatic activity. These observations have consequences with respect to the topology of the N-terminus relative to the active site as well as possible activation mechanisms.
authors.affiliation: Division of Structural Biology, Department of Molecular Biology, University of Salzburg, Billrothstrasse 11, A-5020 Salzburg, Austria.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Eckhard
authors.forename: Esther
authors.initials: E
authors.lastname: Schönauer
authors.forename: Paulina
authors.initials: P
authors.lastname: Ducka
authors.forename: Peter
authors.initials: P
authors.lastname: Briza
authors.forename: Dorota
authors.initials: D
authors.lastname: Nüss
authors.forename: Hans
authors.initials: H
authors.lastname: Brandstetter
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D047009:Hathewaya histolytica / Q000201:enzymology*; D003017:Clostridium tetani / Q000201:enzymology*; D017364:Collagenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005998:Glycine; D006639:Histidine; D007700:Kinetics; D061965:Matrix Metalloproteinase Inhibitors; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D061965:Matrix Metalloproteinase Inhibitors; D010618:Phenanthrolines; D011994:Recombinant Proteins; D006639:Histidine; D017364:Collagenases; D005998:Glycine; C025205:1,10-phenanthroline